Journal: The Journal of Neuroscience

Article Title: Structural and Functional Rescue of Chronic Metabolically Stressed Optic Nerves through Respiration

doi: 10.1523/jneurosci.3652-17.2018

Figure Lengend Snippet: Figure 1. Glycogen and glial glucose transporters decreased with glaucoma pathology. 905 A, Glycogen analysis in 3, 6, and 10-month DBA/2J (D2) and control DBA/2J-Gpnmb+ (D2G) optic nerve (ON), 906 (n=6 ON per group). B and F, Capillary electrophoresis of GLUT1 B, and GLUT3 F, protein in 3, 6, and 10-month 907 D2 and D2G ON normalized to total protein then to 3m D2G protein levels (n=8 ON per group). See Figure 1-1. 908 C, Distribution of GLUT1 in ON of 3m D2, and 10m D2G and D2 optic nerves immunolabeled with GFAP 909 (green). Arrows indicate colocalization of GLUT1 and GFAP, (n=3 sections per ON, 6 ON per group). D and E, 910 Glut1 and Glut3 mRNA levels in 3, 6, and 10-month D2 and D2G ON, normalized to Hprt mRNA then to 3m 911 D2G mRNA (n=5-7 ON per group). See Figure 1-1. G, Distribution of GLUT3 in ON stained with Fluoromyelin. 912 Arrows indicate colocalization of GLUT3 with Fluoromyelin, (n=3 sections per ON, 6 ON per group). H and I, 913 Percent of mean fluorescence intensity in the region of interest (ROI) for C, GLUT1 and G, GLUT3. All values are 914 presented as mean ± SEM, one-way ANOVA and Tukey’s post-hoc test. A, F(5, 30) =18.59, **p = 0.0022, ***p 915 = 0.00011; B, F(5, 42) = 6.463, **p = 0.0077; D, F(5, 30) = 1.906, **p = 0.001; E, F(5, 37) = 15.58, **p= 0.0023, 916 ***p =0.0001; H, F(2, 51) = 210.4, ***p = 0.0001. Scale bar=20μm for C and G. 917 918 Figure 2. Monocarboxylate transporters downregulated with glaucoma pathology. 919 A, Mct1 mRNA levels in 3, 6, and 10-month D2 and D2G ON, normalized to Hprt mRNA then to 3m D2G mRNA 920 (n=5-13 ON per group). See Figure 2-1. B, D and E, MCT1, MCT2 and MCT4 protein levels in 3, 6 and 10-month 921 D2G and D2 ON, normalized to total protein and then to 3m D2G protein levels (n=8 ON per group). C, F and G, 922 Distribution of MCT1 C, MCT2 F, and MCT4 G, in 3m D2, and 10m D2G and D2 ON stained with Fluoromyelin 923 (green) or immunolabeled with GFAP (green). Arrows indicate colocalization (n=3 sections per ON, 6 ON per 924 group). H, Distribution of MCT2 in human control and glaucoma patient ON, immunolabeled for β-tubulin 925 (green) and stained with DAPI (blue). Arrows indicate colocalization (n=4 sections per ON, 2 ON per group). 926 See Figure 2-1. I-L, Percent of mean fluorescence intensity in the ROI for I, MCT1, J, MCT2, K, MCT4 D2 and 927 D2G ON, and L MCT2, human ON. M, Quantification of total number of RGC axons in 6m D2, 10m D2G and 928 10m D2 ON. All values are presented as mean ± SEM, one-way ANOVA and Tukey’s post-hoc test. A, F(5, 47) = 929 4.579, *p = 0.0141; B, F(5, 38) = 6.553, **p = 0.0076; D, F(5, 42) = 16.14, *p = 0.0054, **p = 0.0013; E, F(5, 42) 930 = 4.734, *p= 0.0348; I, F(2, 51) = 173.2, ***p = 0.0001; J, F(2, 51) = 402.1, ***p = 0.0001; K, F(2, 51) = 192.6, 931 ***p = 0.0001; L, t(6) = 9.317, ***p = 0.0001, two-tailed unpaired t-test). Scale bar=20μm applies to C, F, G, 932 and H. 933 934 935 Figure 3. Low lactate accompanies AMPK activation and limits mitochondrial biogenesis and metabolic 936 cofactor pools. 937 A, L-lactate levels in 3, 6 and 10-month D2G and D2 ON (n=8 ON per group). B, Ratio of phosphorylated-AMPK 938 (pAMPK) to AMPK protein in 3, 6 and 10-month D2G and D2 ON (n=8 ON per group). C-D, Phosphorylated 939 AMPK immunofluorescence (magenta) and GFAP (green) micrographs in C, human control and glaucoma ON 940 (n=4 sections per ON, 2 ON per group); and D, 3m D2, and 10m D2G and D2 mice (n= 3 sections per ON, 6 ON 941 per group). Arrows indicate colocalization of pAMPK and GFAP. E-F, Percent of mean fluorescence intensity in 942 the region of interest (ROI) for pAMPK in E, 3m D2, and 10m D2G and D2 mice, and F, human. G-I, Analyses of 943 NAD+/NADH, creatine kinase (CK) activity and PGC1-α levels in 3, 6 and 10-month D2G and D2 mice. G, NAD+ 944 normalized to NADH levels (n=6 ON per group). See Figure 3-1. H, Creatine kinase activity normalized to total 945 protein (n=6 ON per group). I, PGC1-α protein levels normalized to total protein levels and then to 3m D2G 946 protein levels (n=8 ON per group). All values are presented as mean ± SEM, one-way ANOVA and Tukey’s post- 947 hoc test. A, F(5, 42) = 22.04, *p = 0.0124; B, F(5, 42) = 35.51, **p = 0.0032, ***p = 0.0001; E, F(2, 45) = 208.4, 948

Article Snippet: List of antibodies used for IHC and Capillary Electrophoresis analyses 1103 Antibody Dilution RRID Company Host AKT1 1:50 for WES AB_329827 Cell Signaling Technology Rabbit AMPK α1 2B7 1:200 for WES AB_2721834 Novus Biologicals Mouse BDNF 1:100 for IHC AB_630940 Santa Cruz Biotechnology Rabbit Brn3a 1:50 for IHC AB_626765 Santa Cruz Biotechnology Mouse βIII-tubulin 1:1000 for IHC AB_107216 Abcam Chicken GLUT1 1:200 for IHC, 1:50 for WES AB_790014 Novus Biologicals Rabbit GLUT3 1:25 for IHC and WES AB_2191306 R&D Systems Mouse Glutamine Synthetase 1:50 for WES AB_1127501 Santa Cruz Biotechnology Mouse GFAP 1:250 for IHC AB_10917109 EMD Millipore Mouse GFAP 1:500 for IHC AB_10013382 Dako-Agilent Rabbit MCT1 1:25 for IHC and WES AB_10841766 Santa Cruz Biotechnology Mouse MCT2 1:50 for WES AB_2721836 Aviva Systems Biology Rabbit 1:50 for WES AB_10855300 Bioss Rabbit MCT4 1:250 for IHC AB_2254765 Santa Cruz Biotechnology Goat 1:50 for WES AB_2189333 Santa Cruz Biotechnology Rabbit NRF2 1:25 for IHC AB_2618882 Developmental Studies Hybridoma Bank Mouse RBPMS 1:200 for IHC AB_10720427 GeneTex Inc. Rabbit PhosphoAKTSer 473 1:50 for WES AB_329825 Cell Signaling Technology Rabbit pAMPK α1Thr 172 1:100 for IHC, 1:50 for WES AB_11032916 Novus Biologicals Rabbit p70S6K 1:50 for WES AB_331676 Cell Signaling Technology Rabbit Phosphop70S6KThr 389 1:50 for WES AB_330944 Cell Signaling Technology Rabbit Phosphop70S6KSer 411 1:25 for WES AB_2182257 Santa Cruz Biotechnology Mouse SOD2 1:100 for IHC AB_2191814 Santa Cruz Biotechnology Mouse TFAM 1:50 for IHC AB_10610743 Santa Cruz Biotechnology Mouse 1104 48 48 1105 Table 2.

Techniques: Control, Electrophoresis, Immunolabeling, Staining, Fluorescence, Two Tailed Test, Activation Assay, Immunofluorescence, Activity Assay

Journal: eLife

Article Title: Insights into metabolic heterogeneity of colorectal cancer gained from fluorescence lifetime imaging

doi: 10.7554/eLife.94438

Figure Lengend Snippet: ( A ) Representative immunohistochemical images of GLUT3 expression. Scale bar = 50 μm (magnification x200) and 20 μm (magnification x630). ( B ) Representative immunohistochemical images of LDHA expression. Scale bar = 50 μm (magnification x200) and 20 μm (magnification x630). ( С ) Semi-quantitative evaluation of the expression level by staining intensity.

Article Snippet: Next, slides were incubated with primary polyclonal antibodies to Glucose Transporter 3 GLUT3 (E-AB-31557, Elabscience, China) or to Lactate dehydrogenase A LDHA (E-AB-19947, Elabscience, China) for 15 min. For the antibodies detection «Bond polymer refine detection system» (Leica Biosystems, UK) was used.

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: PLoS Pathogens

Article Title: An Oncogenic Virus Promotes Cell Survival and Cellular Transformation by Suppressing Glycolysis

doi: 10.1371/journal.ppat.1005648

Figure Lengend Snippet: ( A-C ). Analysis of GLUT1 and GLUT3 expression in MM and KMM cells by RT-qPCR ( A ), Western-blot ( B ) and flow cytometry ( C ). ( D-F ). Analysis of GLUT1 and GLUT3 expression in cells infected by different recombinant viruses by RT-qPCR ( D ), Western-blot ( E ) and flow cytometry ( F ). β-actin and β-tubulin were used as internal controls for RT-qPCR and Western-blot, respectively. For panels C and F , the Y-axis is shown as normalized cell numbers. All data are presented as mean ± s.e.m. from three (n = 3, A and D ) independent experiments, each with three repeats. Representative images from three independent experiments with similar results are presented ( B, C, E, and F ). NS, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Immunostaining was performed using an anti-LANA antibody LN35 (cat. ab4103, Abcam) and an anti-GLUT1 antibody (ab115730, Abcam) or an anti-GLUT3 antibody (cat. sc-30107, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies, followed by Alexa-488 and Alexa-568 conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Infection, Recombinant

Journal: PLoS Pathogens

Article Title: An Oncogenic Virus Promotes Cell Survival and Cellular Transformation by Suppressing Glycolysis

doi: 10.1371/journal.ppat.1005648

Figure Lengend Snippet: ( A ). Analysis of RelA, GLUT1 and GLUT3 proteins in MM and KMM cells by Western-blot following knock down of RelA. Detection of RelA, GLUT1 and GLUT3 proteins in MM and KMM cells following transfection with a siRNA to RelA (siRelA) or a scrambled control (Scr) for 3 days. β-tubulin was used as an internal control for loading. ( B-C ). Analysis of RelA ( B ), GLUT1 ( C ) and GLUT3 ( D ) mRNAs in MM and KMM cells by RT-qPCR following knock down of RelA. Cells were treated as described in ( A ). β-actin was used as an internal control for qPCR. ( E ). Knock down of RelA decrease cell proliferation of KMM but not MM cells. Cell proliferation were examined following knock down of RelA. ( F-G ) Knock down of RelA increases glucose consumption ( F ) and lactate production ( G ) in KMM cells. Glucose consumption and lactate production were determined as described in following knock down of RelA. Experiments were repeated three times, each with three repeats and representative results were presented. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Immunostaining was performed using an anti-LANA antibody LN35 (cat. ab4103, Abcam) and an anti-GLUT1 antibody (ab115730, Abcam) or an anti-GLUT3 antibody (cat. sc-30107, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies, followed by Alexa-488 and Alexa-568 conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Western Blot, Knockdown, Transfection, Control, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: An Oncogenic Virus Promotes Cell Survival and Cellular Transformation by Suppressing Glycolysis

doi: 10.1371/journal.ppat.1005648

Figure Lengend Snippet: ( A-B ). Inhibition of the NF-κB pathway increases the expression of GLUT1 and GLUT3. MM and KMM cells were treated with NF-κB inhibitors JSH23 (30 μM) or BAY 11–7082 (2 μM), and examined for the expression of GLUT1 and GLUT3 expression by RT-qPCR at 8 h post-treatment ( A ) and flow cytometry at 24 h post-treatment ( B ). Y-axis in ( B ) is shown as normalized cell numbers. β-actin was used as internal controls for qPCR. ( C-D ). Inhibition of the NF-κB pathway increases glucose consumption ( C ) and lactate production ( D ). MM and KMM cells were treated with NF-κB inhibitor JSH23 (30 μM) and glucose consumption and lactate production were determined as described in . ( E-F ). Inhibition of the NF-κB pathway reduces cell proliferation and inhibits cellular transformation. Cell proliferation ( E ) and colony formation in softagar ( F ) were examined in the presence of JSH23 (30 μM) as described in . ( G-H ). Inhibition of the NF-κB pathway increases apoptosis and reduces BrdU incorporation. Apoptosis ( G ) and BrdU incorporation ( H ) of MM and KMM cells were examined following treatment of JSH23 (30 μM) for 48 h as described in . All data are presented as mean ± s.e.m. from three (n = 3, A , and C-H ) independent experiments, each with three repeats. Representative images from three independent experiments with similar results are presented ( B and F ). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Immunostaining was performed using an anti-LANA antibody LN35 (cat. ab4103, Abcam) and an anti-GLUT1 antibody (ab115730, Abcam) or an anti-GLUT3 antibody (cat. sc-30107, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies, followed by Alexa-488 and Alexa-568 conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Flow Cytometry, Transformation Assay, BrdU Incorporation Assay

Journal: PLoS Pathogens

Article Title: An Oncogenic Virus Promotes Cell Survival and Cellular Transformation by Suppressing Glycolysis

doi: 10.1371/journal.ppat.1005648

Figure Lengend Snippet: ( A-B ). Stable expression of GLUT1 and GLUT3 in MM and KMM cells. Cells with stable expression of GLUT1 or GLUT3 were analyzed by Western-blot ( A ) and flow cytometry ( B ). Y-axis in ( B ) is shown as normalized cell numbers. β-tubulin was used as internal controls for Western-blot. ( C-D ). Overexpression of GLUT1 or GLUT3 increases glucose consumption ( C ) and lactate production ( D ). MM and KMM cells with stable expression of GLUT1 or GLUT3 were examined for glucose consumption and lactate production as described in . ( E-G ). Overexpression of GLUT1 or GLUT3 sensitizes KMM cells to glucose deprivation shown in cell proliferation ( E ) and apoptosis ( F ) but not cell cycle distribution ( G ). Cell proliferation, apoptosis and cell cycle progression of MM and KMM cells stably expressing GLUT1 or GLUT3 were examined in the presence or absence of glucose as described in . ( H ). Overexpression of GLUT1 or GLUT3 reduces the efficiency of colony formation of KMM cells in softagar. Colony formation of KMM cells was examined in the presence or absence of glucose as described in . All data are presented as mean ± s.e.m. from three (n = 3, C-H ) independent experiments, each with three repeats. Representative images from three independent experiments with similar results are presented ( A, B, and H ). NS, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Immunostaining was performed using an anti-LANA antibody LN35 (cat. ab4103, Abcam) and an anti-GLUT1 antibody (ab115730, Abcam) or an anti-GLUT3 antibody (cat. sc-30107, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies, followed by Alexa-488 and Alexa-568 conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Expressing, Western Blot, Flow Cytometry, Over Expression, Stable Transfection

Journal: PLoS Pathogens

Article Title: An Oncogenic Virus Promotes Cell Survival and Cellular Transformation by Suppressing Glycolysis

doi: 10.1371/journal.ppat.1005648

Figure Lengend Snippet: ( A ). Effect of GLUT1 and GLUT3 on the AKT-NF-κB pro-survival pathway. Cell lysates from KMM-vector, KMM-GLUT1 and KMM-GLUT3 cells cultured under normal conditions were analyzed by Western-blot with the specified antibodies. β-tubulin was used as internal controls. ( B-D ). Overexpression of GLUT1 or GLUT3 increases autophagy in KMM cells. KMM-vector, KMM-GLUT1 and KMM-GLUT3 cells transduced with mCherry-LC3 for 48 h were examined for the formation of LC3 punctate. Representative images were captured at 1,000x magnification using laser-scanning confocal microscopy ( B ). The percentage of cells with LC3 punctate ( C ) and the number of LC3-positive dots per cell ( D ) were counted. ( E ). Glucose deprivation and inhibition of PI3K reduce NF-κB activation. Total and phosphorylation of NF-κB in KMM-vector, KMM-GLUT1 and KMM-GLUT3 cells in the presence or absence of glucose as well as with and without treatment with 12.5 μM PI3K inhibitor LY294002 were examined by Western-blot at 48 h post-treatment. ( F ). Glucose deprivation and inhibition of PI3K sensitize KMM cells to apoptosis. KMM-vector, KMM-GLUT1 and KMM-GLUT3 cells in the presence or absence of glucose as well as with and without treatment with 12.5 μM PI3K inhibitor LY294002 were examined for apoptosis at 72 h post-treatment. All data are presented as mean ± s.e.m. from three (n = 3, C, D, and F ) independent experiments, each with three repeats. Representative images from three independent experiments with similar results are presented ( A, B, and E ). NS, not significant; *** P < 0.001.

Article Snippet: Immunostaining was performed using an anti-LANA antibody LN35 (cat. ab4103, Abcam) and an anti-GLUT1 antibody (ab115730, Abcam) or an anti-GLUT3 antibody (cat. sc-30107, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies, followed by Alexa-488 and Alexa-568 conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Plasmid Preparation, Cell Culture, Western Blot, Over Expression, Transduction, Confocal Microscopy, Inhibition, Activation Assay, Phospho-proteomics

Journal: PLoS Pathogens

Article Title: An Oncogenic Virus Promotes Cell Survival and Cellular Transformation by Suppressing Glycolysis

doi: 10.1371/journal.ppat.1005648

Figure Lengend Snippet: ( A-B ). Representative illustration of dual immunofluorescence detection of LANA and GLUT1 ( A ) or GLUT3 ( B ) in a normal human skin section and a KS tumor section. The tissue sections were counterstained with DAPI. Magnifications 600x. Boxed areas are enlarged. ( C ) Analysis of GLUT1 expression in LANA negative (-) and LANA positive (+) cells in KS tumors (n = 25). ( D ) Analysis of GLUT3 expression in LANA negative (-) and LANA positive (+) cells in KS tissues (n = 17). ( E-F ) Negative correlation of the average percentage of LANA-positive cells with the average expression level of GLUT1 ( E ) and GLUT3 ( F ) in KS tumors. For ( C and D ), the boxes represent the interquartile range (25-75 th centiles. The horizontal line inside the box indicates the median. The vertical whiskers extend to the maximum and minimum values. Statistical analysis was performed by Wilcoxon matched-pairs signed-ranks test. Expression levels of GLUT1 and GLUT3 were quantified based on immunofluorescence staining, using a modified His-score as described in the Materials and Methods. ** P < 0.01; *** P < 0.001.

Article Snippet: Immunostaining was performed using an anti-LANA antibody LN35 (cat. ab4103, Abcam) and an anti-GLUT1 antibody (ab115730, Abcam) or an anti-GLUT3 antibody (cat. sc-30107, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies, followed by Alexa-488 and Alexa-568 conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Immunofluorescence, Expressing, Staining, Modification

Journal: PLoS Pathogens

Article Title: An Oncogenic Virus Promotes Cell Survival and Cellular Transformation by Suppressing Glycolysis

doi: 10.1371/journal.ppat.1005648

Figure Lengend Snippet: ( A ). Downregulation of GLUT1 in PEL cell lines. Western-blot detection of GLUT1 and GLUT3 protein levels in PEL cell lines BCBL1, BC3 and BCP1, and uninfected and KSHV-infected BJAB cells. ( B ). Cell proliferation rates of PEL cell lines BCBL1, BC3 and BCP1, and uninfected and KSHV-infected BJAB cells. ( C-D ). KSHV-infected cells have reduced levels of glucose consumption ( C ) and lactate production ( D ). Glucose consumption and lactate production were determined as described in . Experiments were repeated three times, each with three repeats and representative results were presented. *** P < 0.001.

Article Snippet: Immunostaining was performed using an anti-LANA antibody LN35 (cat. ab4103, Abcam) and an anti-GLUT1 antibody (ab115730, Abcam) or an anti-GLUT3 antibody (cat. sc-30107, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies, followed by Alexa-488 and Alexa-568 conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Western Blot, Infection

Journal: PLoS Pathogens

Article Title: An Oncogenic Virus Promotes Cell Survival and Cellular Transformation by Suppressing Glycolysis

doi: 10.1371/journal.ppat.1005648

Figure Lengend Snippet: A model illustrates that vFLIP and the miRNA cluster mediate KSHV suppression of aerobic glycolysis to promote cell survival by downregulating the expression of GLUT1 and GLUT3 in an AKT-NF-κB-dependent manner.

Article Snippet: Immunostaining was performed using an anti-LANA antibody LN35 (cat. ab4103, Abcam) and an anti-GLUT1 antibody (ab115730, Abcam) or an anti-GLUT3 antibody (cat. sc-30107, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies, followed by Alexa-488 and Alexa-568 conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Expressing